Electrochemical biosensors have wide practical application in clinical assays, environmental monitoring and process control because they offer excellent sensitivity, fast response, and high selectivity. In addition, they can be easily miniaturized and are inexpensive.
Amperometric response of biosensors for glucose measurement is based on probing of an enzymic reaction either by oxidation of hydrogen peroxide or by the electron exchange from the active center of the redox enzyme. The amperometric signal obtained by the electron exchange from the active center of the enzyme and electrode surface increases the selectivity and sensitivity of analysis considerably since the selective inherent properties of the enzyme are monitored directly. The electron exchange from the active center of an enzyme like glucose oxidase to the electrode surface is facilitated by incorporating electron transfer relays between the active center of the enzyme and electrode surface.
There are a number of reports on the use of organic metals including salts of tetracyanoquinodimethane (TCNQ) as an efficient electrocatalyst for the direct electron exchange from the active center of enzymes. Cenas and Kulys (Bioelectrochem. Bioenerg. 8, 103 (1981)) report that oxidation of redox enzymes at the surface of organic metals proceeds through a mediatory way. They express the current of an enzyme electrode in terms of mediator concentration produced during the slight dissolution of the organic metal salts on the surface of the electrode. Cenas and Kulys (J. Electroanal. Chem. 128, 103 (1981)) have demonstrated that TCNQ can promote electron transfer between glucose oxidase (GOD) and an electrode.
Considering previous works, Hendry and Turner (Horm. Metab. Res. (Suppl.) 20, 37 (1988)) have developed a glucose sensor which utilizes TCNQ as a mediator. However, the sensor suffers from a number of disadvantages including a short half-life of the electrode (i.e., 1-1.5 h), which may be attributed to either loss of enzyme activity, loss of enzyme, or leaching of TCNQ from the electrode, and a limited linear range of the calibration curve (i.e., anodic current is linear up to 20 mM glucose and nonlinear up to 75 mM).
Incorporation of mediators with redox proteins has attracted considerable attention in the development of amperometric enzyme electrodes for glucose. Accordingly, many different classes of redox-active molecules have been characterized as mediators.
Recent reports (Amine et al, Talanta 38, 107 (1991); Hale et al, J. Am. Chem. Soc. 111, 3482 (1989); Matuszewski et al, Analyst 113, 735 (1988)) show that GOD alone, GOD-linked mediator, or GOD and a mediator can be incorporated into a graphite/carbon paste electrode (graphite particles suspended in Nujol oil) (CPE), which results in an enzyme electrode with enhanced mechanical and electrochemical stability. The extended linearity of the sensor resulting is attributed to diffusion-limited conditions through and within the oily electrode interface. However, the limited electrode stability (8 days) is related to progressive leaching out of the mediator from the electrode (Amine et al, Talanta 38, 107 (1991).
Amperometric biosensors have been employed in batch type reactor systems for detecting glucose. However, flow-injection analysis (FIA) for the simultaneous determination of several components in very small sample volumes has wider application, because flow-injection systems allow well-defined and highly reproducible concentration transient at detector sites and can be operated in a variety of flow-injection modes.
Several flow-injection analysis systems which incorporate immobilized GOD for the analysis of glucose based on electrochemical detection and chemiluminometric determination are known. The electrochemical mode of detection in such systems is based on the measurement of electron-exchange from the anodic oxidation of hydrogen peroxide produced by the enzymatic reaction of GOD.
Recently a flow-injection analysis system for the on-line measurement of glucose, glutamate, and lactate in brain microdialysate has been described (Boutelle et al, Anal. Chem., 64, 1790 (1992). In this system the enzyme horseradish peroxidase is regenerated by the oxidation of two ferrocene species present in a buffer which is pumped through a packed bed.
The present invention is directed to a flow-injection analysis (FIA) system which incorporates a biosensor for glucose which provides a wide linear dynamic range and good mechanical and electrochemical stability.